Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures  High-resolution structures of proteins are essential for understanding cellular processes. Determining protein structures, however, is difficult: protein stability, purity, quantity, and solubility critically affect success. Nuclear magnetic resonance (NMR)We present a novel approach to obtain structural details of proteins by mass spectrometry. This can be accomplished through cross-linking and mass spectrometry (CLMS) –11. Crose.g. the proteasome . Human blood serum was acquired from a healthy male donor after informed consent, in accordance with standard institutional ethical procedures at the University of Edinburgh, School of Biological Sciences. Immediately following collection , blood serum was isolated from the whole blood sample without anti-coagulants, by centrifugation. Whole blood was allowed to clot by leaving it undisturbed at room temperature for 30 min. The clot was removed by centrifuging at 1900 × m) in cross-linking buffer  to initiate incomplete lysine reaction with the sulfo-NHS ester component of the cross-linker. The diazirine group was then photo-activated using UV irradiation. A UVP B-100AP, 100 W mercury lamp at 365 nm was utilized for photo-activation. Samples were spread onto the inside of Eppendorf tube lids to form a thin film, placed on ice at a distance of 5 cm from the lamp and irradiated for either 1, 10, 20, 30, 40, 45, or 60 min. The resulting cross-linked mixture was separated on a NuPAGE 4–12% Bis-Tris gel using MES running buffer and Coomassie blue stain.Mixing ratios of sulfo-SDA to HSA were titrated using cross-linker-to-protein weight-to-weight ratios of 0.25:1, 0.5:1, 1:1, 2:1, 4:1, and 8:1. Either purified HSA or whole blood serum  was mixed with sulfo-SDA  in the ion trap. 1+ and 2+ ions were excluded from fragmentation. Fragmentation (MS2) spectra were acquired in the Orbitrap at 7500 resolution. Dynamic exclusion was enabled with 90 s exclusion time and repeat count equal to 1.Peptides were loaded directly onto a spray emitter analytical column  packed with C18 material  using an air pressure pump (Proxeon Biosystems) . Mobile Mass spectrometric raw files were processed into peak lists using MaxQuant version 1.2.2.5  using desupplemental Tables S1–S4. Cross-links were visualized in the crystal structure of HSA (PDB 1AO6) (Peak lists were searched first against the human subset of UniProt (ipi.HUMAN.v3.79) using Mascot (version 2.4.0) and search parameters: MS accuracy, 6 ppm; MS/MS accuracy, 20 ppm; enzyme, trypsin; specificity, fully tryptic; allowed number of missed cleavages, two; fixed modifications, none; variable modifications, carbamidomethylation on cysteine, oxidation on methionine. This revealed that HSA was by far the most abundant protein in our bands, in both purified and human blood serum samples. Peak lists were subsequently searched against two databases using Xi  for identification of cross-linked peptides. One database contained all proteins identified in the initial Mascot search, the other contained only the sequence of HSA (UniProt P02768). Search parameters were MS accuracy, 6 ppm; MS/MS accuracy, 20 ppm; enzyme, trypsin; specificity, fully tryptic; allowed number of missed cleavages, four; cross-linker, SDA; fixed modifications, none; variable modifications, carbamidomethylation on cysteine, oxidation on methionine, SDA-loop . The linkage specificity for sulfo-SDA was assumed to be at lysine, serine, threonine, tyrosine and protein N termini at one end, with the other end having specificity for any amino acid residue. False discovery rates (FDR) were estimated following a modified target-decoy search strategy , 26. In DB 1AO6)  using PyDB 1AO6) .1AO6) is comprised of 576 residues. Proteins of this size are challenging for conformational space search as the search space grows exponentially with protein length. To demonstrate the feasibility of our combined CLMS/search approach, we decided to split HSA into smaller domains more suitable for state-of-the-art protocols. We employed the following computational domain boundary prediction methods: DoBo  constraints using a modification of our previously described model-based search (MBS) . Model-bMBS operates by generating a set of candidate structures based on a small number of Rosetta's Monte-Carlo runs. The resulting structures are clustered by a heuristic clustering procedure to identify densely sampled low-energy regions. We interpret these regions to represent funnels in the energy landscape. MBS then judges the quality of a funnel by refining the five lowest-energy structures in an all-atom force field. The different funnels, along with their estimated quality, form an approximate model of the energy landscape. In subsequent iterations, MBS reallocates computational resources from low-quality funnels to promising regions of the conformational space.AbInitioRelax protocol. In the following 4 MBS stages, the conformational space is searched by 9-mer fragment replacements (Rosetta stage 3). The resulting structures are refined in the final MBS stage by 3-mer fragment replacements (Rosetta stage 4). Each stage of MBS is interleaved with a clustering step to determine an approximate model of the energy landscape and to reallocate computational resources, as described above. We generate 5000 structures in each stage of MBS.MBS consists of six stages. Stage 1 builds coarse topologies, using stages 1 and 2 of Rosetta's et al. (The three HSA domains contain long loops and we accounted for their high flexibility in modeling by the following treatment: We explicitly modeled the long loop regions (longer than 15 amino acids) predicted by DISOPRED2 , but remet al. . The samC). To model these constraints as part of the energy function during conformational space search, we use a modified Lorentz function:ijd is the distance between residues i and j in the structure, the lower bound l = 1.5Å and the upper bound u = 20Å are distance limits within which the maximum energy bonus (maxE) is rewarded. The parameter w = 1.0 regulates how quickly the energy bonus decreases when dij is not within the lower/upper bounds, with w being the half-width i.e. the violation in Å where maxE/2 is still rewarded.For CLMS constraints obtained by SDA, we used 20Å as the upper bound for the distance between solvent-accessible amino acids. We set the upper bound to 20Å, the maximum through-space Cα-Cα distance of amino acids with long side-chains (like lysine and arginine) plus the sulfo-SDA linker distance and some added distance to allow for conformational flexibility. This is a conservative estimation of the upper distance bound of the SDA cross-linker and agrees with the experimentally observed upper limits when comparing CLMS data with the crystal structure of HSA see C. To modl<dij≤u), the Lorentz term will award an energy bonus Emax. If the constraint is mildly violated dij-l ≤ 2w or u-dij ≤ 2w, the energy bonus decreases. However, if the constraint is significantly violated (l-dij>2w or dij-u>2w), the influence of the constraint will approach zero, effectively ignoring the constraint in the energy calculation. Intuitively, the resulting energy term attempts to maximize the number of constraint satisfied by a structure, rather than the exact distances.If a constraint is satisfied  using MBS and 320/107/248 CLMS distance constraints for domains A/B/C for purified HSA in solution and 248/68/163 CLMS constraints for HSA in blood serum. Note that we only used CLMS constraints with a sequence separation larger than 12aa. These longer constraints are more informative than short-range constraints. The latter mainly carry spatial information from secondary structure elements.We employ CLMS constraints together with predicted contacts to guide search during all low-resolution stages of MBS. We use CLMS constraints at 20% FDR. We predict the structure of the individual domains of HSA . This combined information enables effective conformational space search. Our results serve as a preliminary indication that the synergistic combination of experiment and computation may lead to efficient, cost-effective, high-throughput structure determination methods.We tested several methods for structure selection in their ability to select a single structure (first structure), and a low-RMSD structure within a small number (we choose five in this study) of top ranked structures (best structure). We tested Rosetta's physically realistic all-atom energy function, clustering with Durandal , Lorentzsupplemental Fig. S1). This group is stable under normal conditions but can be activated by light (320–370 nm) to generate a highly reactive carbene , it has a known crystal structure and is commercially available at a low cost in a purified form. HSA is also the most abundant protein in human blood plasma, making up more than half the total protein content. One of the technical challenges for CLMS is the mass spectrometric detection of cross-links, which is easier in low rather than high complex mixtures. Enriching for the target protein following the cross-linking step reduces mixture complexity and enhances cross-link detection. Also other structure determination approaches, NMR and x-ray crystallography, require protein purification. Notably, the purification requirements are large for these established approaches with regards to protein amount and purity. Furthermore, they require the protein to be native throughout the enrichment as the structure is subsequently analyzed. CLMS fixes the structure first, allowing the enrichment to employ procedures that would otherwise disrupt protein structure, Asupplemental Fig. S2). Proteomics generally suffers from stochastic detection of analytes especially when these are present at low levels. This is not unique to cross-links but also affects linear peptides albeit to a much lesser extent owing to their generally higher intensity . For linear peptides a single run essentially returns 50% of the identifications under our conditions that focused on cross-linked peptides (singly and doubly charged precursors excluded). As we discuss later, the high number of constraints at 10 and 20% FDR has larger value for protein structure modeling than the increased confidence at lower FDR. Thus we focus our results discussion on CLMS data at these FDR rates . The majority of the observed constraints in blood serum HSA are in agreement with those of purified HSA . supplemental Tables S1 and S3 contain the peptide-spectrum matches and supplemental Tables S2 and S4 contain all cross-links from this work for up to 20% FDR, along with a confidence score.We cross-linked purified HSA and human blood serum using sulfo-SDA in a two-step procedure. First, protein was labeled by sulfo-SDA in the dark and then the labeled protein was exposed to UV light. The cross-linked protein was subjected to PAGE, HSA excised and subsequently digested and analyzed by LC-MS using a high-high acquisition strategy . Using tDR rates . We idenA cross-linked peptide comprises two peptides and a linkage site within each. NHS-esters have limited reactivity, which helps in assigning linkage sites. The total promiscuity of the carbenes resulting from photoactivated diazirine means that the diazirine cross-linking end can theoretically be placed at any residue within a peptide. For accurate site assignment one requires backbone fragmentation on either side of a linkage site. We manually checked for the supporting evidence of the linkage sites returned by our search, for a random subset of 78 unique cross-links and 368 supporting spectra with wide score distribution. We found that in 38/78 (49%) of the unique cross-links and 77/368 (21%) of matching spectra, the diazirine linkage site was supported by at least one fragmentation event on either side of the assigned site. In cases where there is no fragmentation evidence to pinpoint the exact site of diazirine reaction, our algorithm places the linkage site on the first amino acid within the region indicated by flanking fragmentation events. Some of these sites will be wrong and future implementations should report supported regions rather than imprecise sites. Site imprecision in addition to false sites are therefore errors that need to be considered during protein modeling.supplemental Fig. S4). The randomization was done by locally shifting all (diazirine) sites randomly once within a window of residues. We considered window sizes from 3 to 39 residues and all residue pairs that served as input in our protein modeling . Assuming p = 0.05 to be the significance threshold, distance distributions differed significantly only when considering a window of 11 residues. This means that our site assignment could have erred within ± 5 residues around our called site before the resulting distance distributions would have been significantly different. However, this does not mean that at this point the modeling would have been affected. The median shift of the distribution is very small; at window size 11 the distribution shifts by 1.00 ± 0.16 Å and even at window size 39 it does not shift more than 4 Å. Note that the median length of our diazirine cross-linked peptides is 12 residues. Consequently, identifying the peptide suffices and calling a random residue within this peptide as linkage site hardly impacts on the distance distribution.We gave consideration to testing the possible impact of imprecise site calling on our constraint data quality. For this, we compared our distance distribution of linked residue pairs with a partially randomized data set  , albeit modeling . Earliermodeling , 19, whiTypically, searching the conformational space for the native structure is difficult because of the size of the space and the ruggedness of the energy landscape, although for small proteins this has been achieved . Howeversupplemental Table S5). Domains of purified HSA have the following sizes and number of CLMS constraints: domain A: 197 amino acids, 22.6 kDa, 320 CLMS distance constraints; domain B: 189 amino acids, 21.5 kDa, 107 distance constraints; domain C: 192 amino acids, 21.7 kDa, 248 distance constraints, we only used CLMS constraints with a sequence separation of 12 or more residues. CLMS data leads to increased sampling of near-native conformations compared with sampling without constraints (A–5C). The structures determined for domains A/B/C of purified HSA have an RMSD of 2.8/5.6/2.9 Å to the crystal structure (PDB 1AO6) . We mostly find deviations from the crystal structure in the long loop region, probably because of its inherent flexibility, and in the interface regions between domains. However, the low energy ensembles display good convergence and sampling around the native structure . The relatively high RMSD of the model of domain B is the result of a reduced number of available CLMS distance constraints for this domain.Using this procedure, we generated structures of the three domains of HSA using the CLMS constraints at 20% FDR from purified HSA and from HSA samples in serum. The division of HSA into domains is necessary, as existing computational methods cannot yet address the size of proteins that can be analyzed experimentally . In addition, we tested the impact of BS3 cross-linking data on structure modeling success and found BS3 information insufficient to aid the modeling process (RMSD 10.2/11.9/10.4 Å for the best structure among the ten lowest-energy structures) .Without the use of CLMS constraints, the RMSDs for the best models obtainable by conformational space search alone have low resolution (RMSD 7.9/7.4/15.5 Å for the best structure among the ten lowest-energy structures) . Overall, MBS samples the lowest RMSD structures with CLMS data at 10 or 20% FDR. Except for domain A (purified), 20% FDR yields the highest ensemble quality, with the RMSD at the 1% percentile dropping from 4.1/5.3/6.4 Å (1% FDR) to 3.6/4.2/3.1 Å (20% FDR), for domains A/B/C. Thus, a high number of CLMS constraints, even at significant degrees of noise (10–20%), is more effective in modeling HSA domains than few, accurate links at low FDR (1–5%). This is especially true for domain C: The average backbone quality of the sampled structures improves dramatically from 6.4 at 1% FDR to 3.1 Å at 20% FDR.We also tested the impact of the FDR on the backbone quality of the structure ensemble to investigate the tradeoff between cross-link accuracy and quantity. We repeated the MBS calculations five times with CLMS data at 1/5/10/20% FDR and quantified the ensemble quality by the RMSD at the 1% percentile . The combination of Rosetta Energy and CLMS constraints is able to consistently select a single structure with correct topology  from the structure ensemble . Rosetta energy in combination with an orientation-dependent all-atom statistical potential (GOAP) performs best for HSA domains (2.5/4.9/2.9 Å for domains A/B/C). The best-out-of- five structures selected by Rosetta energy+GOAP are closer to native than the first structures selected by Rosetta energy+CLMS constraints . The structure of domain B shows larger deviations from native (RMSD 5.9 Å), because of a smaller number of CLMS constraints (248/68/163 constraints for domains A/B/C) , but still coarsely resembles native topology. The best of five structures are in close agreement with the native structure .We repeated the modeling experiments on the HSA domains with CLMS data from serum samples to test the ability of the approach to probe the structure of HSA in its native, biological environment. Once cross-linking has taken place, a protein can be isolated without concerns about its structural integrity. We tested this by cross-linking HSA in serum and then enriching HSA by running SDS-PAGE . The RMSFig. 5Interestingly, slightly better first structures can be selected with CLMS data from purified HSA than with CLMS data from serum samples (RMSD improvement from (4.5/5.9/4.8 to 3.6/5.9/4.5 Å). Although this would not be possible in a real application in complex environments, this is another indication that CLMS data is valuable for conformational space search and structure selection.We presented an approach that combines experimental high density data from photo-CLMS with conformational space search to recapitulate the structure of human serum albumin (HSA) domains in solution. We also showed that by combining high density data from photo-CLMS with computational biology we are able to study the structure of HSA domains in a complex mixture of proteins, human blood serum. HSA is the most abundant protein of human blood serum, a fact that largely simplified the enrichment of the protein after the cross-linking reaction. Less abundant proteins will require a more elaborate enrichment than simply running an SDS-PAGE, as was done here. However, our proof-of-concept success with HSA suggests the possibility that photo-CLMS and computational biology will reveal the structure of other proteins in their native environment. Prior to routine application, our approach now needs to be optimized and improved in many directions including chemistry, sample preparation, mass spectrometric acquisition, reducing the number of required acquisitions, data analysis and use of constraints during modeling. Ultimately, we envision photo-CLMS and conformational space search will be an experimentally simple and cost-effective complement to established structure determination methods, NMR and x-ray crystallography.


Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated. •Single-molecule FRET reveals previously hidden conformations of AnkyrinR•We show that ankyrin repeats of AnkyrinR switch between high- and low-FRET states•Switching is controlled by an unstructured “safety pin” from an adjacent subdomain•The results suggest that the order-disorder transition regulates AnkyrinR activity Using single-molecule FRET, Tsytlonok et al. reveal previously hidden conformations of AnkyrinR. The ankyrin repeats of AnkyrinR switch between two FRET states, and the switching is controlled by an unstructured “safety pin” from an adjacent subdomain. Therefore, the order-disorder transition regulates AnkyrinR activity. Single-molecule measurements can resolve the complexity that is inherent in protein energy landscapes but obscured in ensemble experiments because of sample averaging. Such studies of small single-domain proteins and miniproteins have yielded fundamental insights into folding mechanisms . In otheD34 is a 426-residue 12-ankyrin-repeat fragment from the giant multidomain protein AnkyrinR  Figure A, one ofPrevious ensemble experiments showed that D34 unfolds via an intermediate state in which the six C-terminal ankyrin repeats are folded and the six N-terminal ankyrin repeats are unfolded . The datD34 comprises residues 402–827 from AnkyrinR, corresponding to the 12 C-terminal ankyrin repeats of the 24-ankyrin-repeat membrane-binding domain and an unstructured loop from the SBD that folds back onto the ankyrin repeats A. It has2 = 2/3) is assumed. We then confirmed this assumption by measuring the fluorescence anisotropies for each double-cysteine variant and all labeled single-cysteine variants /FRET to probe the conformations of ANK3-5, ANK5-8, ANK8-12, and ANK9-12. In TCCD experiments, two (blue and red) overlapping laser beams simultaneously excited a dilute (50 pM) solution of the D34 molecules as they diffused through the confocal volume one molecule at a time. Donor and acceptor fluorescence bursts are detected in coincidence when they originate from the same molecule, therefore allowing direct quantification of the number of molecules with both donor and acceptor fluorophores on them. In FRET, the donor fluorophores were excited with only the blue laser to measure the separation between the dye pairs because the measured simultaneous bursts depend on the conformation of an individual species as it passes through the confocal volume.Data analysis for both TCCD and FRET measurements was carried out using a coincidence criterion described in detail previously . The datA/ID), where IA and ID are the acceptor and donor fluorescence intensities, respectively, having been corrected to account for background and crosstalk. The Z parameter histograms for FRET and TCCD measurements of ANK3-5 are shown in smFRET experiments were performed first under native conditions (50 mM Tris-HCl buffer (pH 8), 150 mM NaCl, 1 mM EDTA, and 0.001% Tween20). The FRET efficiency histogram for wild-type D34 (ANK3-5) is shown in The FRET efficiency histogram of ANK5-8 shows a broad distribution of events peaking at a low efficiency of ∼0.4 B. This rThe two cysteine residues of ANK9-12 are in close proximity (25 Å), like those of ANK3-5. However, the histograms obtained for ANK9-12 are very different from those for ANK3-5 B. The FR2SO4 has been found to stabilize the compact states of proteins .Kosmotrope Nans e.g., . We therSingle-molecule urea-induced equilibrium unfolding experiments were performed next see  and is a2SO4  that is packed against the five most C-terminal ankyrin repeats of D34 in the crystal structure A. To inv ANK9-12 . These rWe used single-molecule FRET to systematically probe the energy landscape of D34. To quantify the changes in the populations of the different FRET states at increasing denaturant concentrations, we used both TCCD with two-laser excitation of the samples and smFRET using only single-laser (blue) excitation. Using the TCCD method, we found that the total number of molecules was constant across the range of urea concentrations, whereas this number decreased gradually with increasing urea concentration when smFRET measurement was used. These results underline the importance of using the two techniques in combination to fully understand the biophysical properties of a protein because TCCD permits the assessment of the number of dual-labeled molecules, even those showing very low FRET.Previous ensemble analysis showed that the unfolding of D34 is a complex and multistep process in which the unfolding of the N-terminal subdomain occurs cooperatively, whereas the C-terminal subdomain unravels in a noncooperative manner . MoreoveAnk genes are subject to varied and tissue-specific mRNA splicing events that result in heterogeneous populations of Ankyrin polypeptides  measurements were performed using a homebuilt laser confocal microscope. The instrumentation, data acquisition, and data analysis have been described in detail previously, including validation of the TCCD/FRET measurements by PAX , and areL.S.I. and D.K. conceived the research. M.T., S.M.I., P.J.E.R., W.X., and M.J.R. performed research and analyzed data. M.T., S.M.I., A.O., D.K., and L.S.I. wrote the paper.


Little is known about the effects of chronic fluoxetine on 5-HT transmission in the adolescent brain, even though it is acknowledged that the neuroplasticity of the brain during childhood and adolescence might influence the neurobiological mechanisms underlying treatment response. Also, possible ongoing effects on monoamine function following drug discontinuation are unidentified. We therefore examined the chronic effects of fluoxetine on extracellular 5-HT and dopamine concentrations in the medial prefrontal cortex and studied their responsiveness to an acute 5-HT challenge after a one-week washout period, both in adolescent and adult rats. Noradrenaline was measured in adult animals only. Fluoxetine increased 5-HT to 200–300% of control and DA and NA to 150% of control. Although there were no lasting effects of chronic fluoxetine on basal monoamine levels, we observed a clear potentiating effect of previous treatment on the fluoxetine-induced increase in extracellular 5-HT and, to a lesser extent, extracellular DA. No differential effect was found for noradrenaline. Age-at-treatment did not influence these results. So, after cessation of chronic fluoxetine treatment 5-HT responsiveness remains heightened. This may be indicative of the continuing presence of 5-HT receptor desensitization, at least until one week after drug discontinuation in rats. No apparent age-at-treatment effects on extracellular monoamine concentrations in the medial prefrontal cortex were detected, but age-related differences in 5-HT transmission further down-stream or in the recovery processes cannot be ruled out. The first onset of mood- and anxiety disorders usually occurs during (early) adolescence. It has therefore been suggested that early treatment of these disorders needs to focus more on youth. At the same time, in order to unravel its underlying mechanisms, research should lay more emphasis on the developing brain instead of the adult brain Preclinical studies investigating the effects of antidepressant drugs on the developing brain still are in short supply, although several lines of research suggest that SSRIs have the potency to alter neurotransmitter development through their effect on 5-HT transmission and to affect consequently also neuronal outgrowth and maturation Extracellular 5-HT concentrations reflect changes in 5-HT activity and increases in these concentrations are known to modulate the MRI signal in the rat brain The objective of this study was to determine if there are ongoing effects of chronic FLX after drug discontinuation on extracellular monoamine concentrations in adult- and adolescent-treated rats. Our aim was twofold; on one hand we wanted to gain more knowledge on the long-term effects of FLX on extracellular monoamine concentrations and on the other hand we liked to know if these long-lasting effects differ in relation to the age-of-treatment. To that end, adolescent and adult rats were chronically treated with FLX and, after a one-week washout period, extracellular 5-HT concentrations in the medial prefrontal cortex (mPFC) were measured before and after an acute FLX challenge using microdialysis. The acute challenge should significantly elevate the levels of 5-HT, DA, and noradrenaline (NA) in the mPFC All experiments were carried out in accordance with the Dutch regulations governing animal welfare and protection. Protocols were reviewed and consented by the Committee on the Ethics of Animal Experiments of the Academic Medical Center of Amsterdam (registered under project number: DDR102588), in accordance with the guidelines of the European Communities Council Directive of 24 November 1986 (86/609/EEC). All invasive procedures were performed under anesthesia, and all efforts were made to minimize suffering and to limit the total amount of animals used.2)), under standard housing conditions and a 12 hour light-dark cycle. Food and water were available ad libitum. Water intake and bodyweight were monitored on a daily base.Male Wistar rats (N = 32) were obtained from Charles River Inc. (Germany). Age of arrival was either post-natal day 21  or PND61 . Within each age category, there was one experimental and one control group (n = 8). All rats were group-housed during treatment, until stereotactic surgery . PND25 was selected because it approximates the start of adolescence in humans. In male rats, adolescence lasts from PND28 to PND60 On the day of surgery (3 days post-treatment), the rats were anesthetized with isoflurane  with a total flow of 0.3 L/min). The animals were then placed in a Kopf stereotactic apparatus  and kept at a constant temperature using a heating pad. A small incision was made to expose the skull after which lidocaine was used for local anesthesia. A microdialysis guide  was placed just above the mPFC  and individually corrected for distance IAP-Bregma: ∼12.72 mm; Medial-Lateral: 0.3 mm; Dorsal-ventral from skull: –3.0 mm (adolescent-treated rats) or −4.0 mm (adult-treated rats), according to Paxinos and Watson 1986). A cannula was placed into the right jugular vein, for intravenous administration of the FLX challenge, and tunneled subcutaneous to the skull were it was attached to a stainless steel 20 G curved blunted needle. The jugular vein cannula was filled with a solution of polyvinylpyrrolidone-amoxycycline-heparine , 0.1 mL (5000 IU/mL)). The probe and jugular vein cannula were fixed to the skull surface with three stainless steel screws and dental glasionomere cement . Rats were given subcutaneous Metacam  post-operatively for pain management and inhibition of inflammation. After surgery, the animals were individually housed.After a recovery period of 5–6 days, microdialysis experiments were performed during the light cycle (8 or 9 days post-treatment). Experiments were carried out in a separate room with an experimental setup suited for four rats. The setup consisted of four standalone Raturn BASi turntables and corresponding counter-balanced lever arms , allowing free movement of the animal during microdialysis. Each setup was equipped with several lengths of Peek-tubing (1.2 µL/10 cm), a microdialysis pump  and a fraction collector . To prevent probe-ultrafiltration, ringer solution was not only pushed but also pulled out of the tubing by the same pump. The microdialysis probes had an exposed membrane of 3 mm (adolescent-treated) or 2 mm . Probes were perfused with Ringer solution  at a constant flow rate of 1 µL/min beforehand overnight. Early in the morning, rats were transferred to the test room and connected to the setup by tether and peek tubing. Perfusion was kept on for approximately 6 h and samples were collected every 15 min in vials containing 5 µL of antioxidant agent  en 0.01% cysteine.HCl ). After an acclimatization period of approximately two hours and one and a half hour of baseline sampling, an acute challenge with FLX  was given intravenously through the jugular vein cannula. After another one and a half hour of sampling, tetrodotoxin  was administered via the perfusion medium through the microdialysis probe into the mPFC for the duration of one hour, in order to inhibit neuronal monoamine release. This resulted in six basal level samples, six samples following the acute FLX challenge and four samples following the TTX challenge. All samples were stored at minus 20°C until analysis.The monoamines 5-HT, DA, and NA and their metabolites  were analyzed using a high performance liquid chromatography (HPLC) ALEXYS 100 2D system equipped with electrochemical detection (DECADE II) from ANTEC Leyden . Samples of 15 µL were spliced in 2 parts of 5 µL and injected on two ALF-115  columns to analyze dopamine and serotonin and their metabolites. The mobile phase for the ALF-115 columns consisted of 100 mM phosphoric acid, 1 mM KCl, 2.43 mM octanesulphonic acid (OSA) and 16% methanol in milliQ water adjusted to a pH of 3.30. The flow rate was kept constant at 0.035 mL/min. Separation was performed at 38°C and the electrochemical potentials were set at 600 mV against an Ag/AgCl reference in the ISAAC electrochemical cell. The signals were analyzed using Clarity (3.0.3.358) software. The detection limits in a 5 µL sample  were 0.02 nM for the monoamines and DOPAC, 5HIAA and 0.2 nM for HVA.2/O2 intoxication. The brains were then removed and snap frozen in isopentane on dry-ice and stored at −20°C until histological examination. Coronal sections of 35 µm were cut on a cryostat system and stained with cresyl violet for verification of the probe placement.After completion of the experiment, all rats were decapitated after deep COFLX and NFLX were analyzed in serum using LC-MS/MS in the positive ionization mode on a Thermo Scientific  Surveyor LC coupled to a Thermo Scientific Quantum Access MS. To 100 µl of serum sample, 250 µl acetonitril/methanol 84∶16 (v/v) containing 400 ng/mL of the internal standard doxepin, was added to precipitate proteins. Samples were vortexed, stored at −20°C for 30 minutes, vortexed again and centrifuged. Of the supernatant, 10 µl was injected onto a Thermo Scientific HyPurity Aquastar  column. A stepwise chromatographic gradient was applied using acetonitril, water and a constant buffer concentration of 0.2% (v/v) ammonium acetate/0.02% (v/v) trifluoracetic acid at pH 3.5. The flow was 0.4 ml/min and the column-oven temperature was 30°C, giving a total chromatographic runtime of 3 minutes per sample, with FLX and NFLX eluting after 2.4 and 2.3 minutes respectively. FLX, NFLX and the internal standard doxepin were measured using the mass transitions 310.1/148.1, 296.1/134.1 and 280.2/107.1 respectively. The method was validated over a range of 20.0–800.0 ng/mL for both compounds. For FLX, the accuracies ranged from 91.5% to 107.8%, the intra-day precisions were below 10.3% and the inter-day precisions were below 17.6%. For NFLX, the accuracies ranged from 93.4% to 109.8%, the intra-day precisions were below 9.2% and the inter-day precisions were below 14.8%.p<0.05. All statistical analyses were performed in SPSS (IBM SPSS Statistics v19.0).Baseline values of extracellular monoamine concentrations were defined as the mean of the five samples before the FLX challenge. All data were then converted to a percentage of that baseline (set as a 100%). All data are presented as mean ± S.E.M. and statistically analyzed using two-way repeated measures analysis of variance (RM-ANOVA). In case of violations of sphericity, Greenhouse-Geisser correction was used. In addition, the AUC-value was determined  for each rat. The effects of age at time of treatment and type of treatment was assessed by using age and treatment as the ‘between subjects’ factors and time as the ‘within subjects’ factor. Effects of treatment on bodyweight and water intake were also taken into account. The criterion for significance was set at Correct placement of the microdialysis probe in the mPFC was confirmed for all animals. For a graphical overview of probe placement and a representative histological example we refer to 23,322 = 17.46, p-value<0.001)). At the end of treatment, FLX-treated animals weighted 378.0 g (±12.8) whereas the adult vehicle-treated animals weighted 391.1 g (±7.2). This difference in bodyweight was no longer present at time of surgery or at time of the microdialysis experiments (average weight 403.1 g±11.0).For both the adult and the adolescent groups, there was no significant difference in bodyweight between the FLX-treated and vehicle-treated animals at the start of treatment . In the adolescent-treated animals, there was no significant effect of FLX treatment on weight gain. There were no significant differences in bodyweight between the FLX-treated and vehicle-treated animals at the end of treatment, at the time of surgery or at the time of the microdialysis experiments (average weight 252.5 g±17.0). In the adult-treated animals, there was a small but significant effect of treatment on weight gain . This is most probably due to the taste of the drinking water being affected by FLX and the concentration of FLX in the drinking water being higher in the adults. Since no major weight loss (over 10% weight loss or less weight gained compared to controls) was observed at any time point, we believe that water intake was sufficient for all animals.For both the adult and the adolescent groups, there was no difference in water intake between the FLX-treated and vehicle-treated animals during the acclimatization period, thus before start of treatment. Relative water intake was significantly higher in younger animals than in the adult animals  and water intake also significantly decreased with aging in the young animals. There was a clear effect of FLX on water intake, especially in the adult animals. In young animals, water intake was on average 8.7% lower in the FLX-treated animals (n.s.). In the adult groups, this difference was 30.5% . Total plasma concentration of active components (FLX plus NFLX) on day 18 of treatment was (on average) 262.13 ng/mL (±49.0) for the adult-treated animals and 124.67 ng/mL (±9.6) for the adolescent-treated animals. Plasma concentrations of FLX alone were below the official detection limit of 20 ng/mL for all adolescent-treated animals and just above or around 20 ng/mL for most of the adult-treated animals.On average, the adult animals received a dosage of 131.1 mg/l FLX hydrochloride per day. With an average daily water intake of 0.041 mL/g, this resulted in an average consumption of 5.31 mg/kg (±0.21) FLX hydrochloride per day. The adolescent animals received an average dosage of 52.8 mg/l hydrofluoxetine. With a daily average water intake of 0.097 mL/g, this resulted in an average consumption of 5.03 mg/Kg (±0.42) FLX hydrochloride per day for the adolescent-treated animals. There was no significant difference in drug intake between the two age groups . On average, the basal level of extracellular 5-HT in the mPFC was 0.170 nM (±0.067). The acute i.v. FLX challenge had a significantly increasing effect on 5-HT release compared to baseline values in all groups ; see also 10,230 = 4.417, p-value = 0.007) as well as within the AUC-values . The increase in 5-HT concentrations compared to baseline was on average 197% in the vehicle-treated animals, while it was 293% in the FLX-treated animals. There were no age or age-by-treatment effects, neither in the repeated measures , nor in the AUC-data . Since the levels of 5HIAA were out of range for several animals, changes in 5HIAA concentrations could not be determined. Therefore, 5-HIAA levels were not analyzed.Basal levels of 5-HT did not differ between groups; there were no statistically significant effects of age, treatment or age-by-treatment . On average, the basal level of extracellular DA in the mPFC was 0.168 nM (±0.110). The acute FLX challenge had a significantly increasing effect on DA release compared to baseline values in all groups ; see also 1,23 = 4.914, p-value = 0.039), but not within the repeated-measures analysis ; see 1,19 = 1.117; p-value = 0.304; age x treatment: F1,19 = 2.912; p-value = 0.104), nor in the AUC-data . Although the FLX treated animals tended to have both higher baseline DOPAC and HVA values, there were no significant effects of either treatment or age for both DA metabolites . The average basal amount of DOPAC was 6,601 nM  and 7,900 nM  for HVA. There was no significant effect of the acute challenge on either DOPAC or HVA values in any of the groups. Average DOPAC and HVA levels after the challenge were 6,641 nM  and 6,903 nM , respectively.In four adult-treated animals, DA values were not quantifiable. Therefore, both the adult FLX-treated and the adult vehicle-treated group consisted of six animals in the DA analysis. Basal levels of DA did not differ between groups; there were no statistically significant effects of age, treatment or age-by-treatment ; average increase of 170%). The basal level of extracellular NA was on average 0.226 nM (±0.154)  had reduced extracellular 5-HT in the frontal cortex to 91% of the baseline values . Extracellular levels of DA and NA were reduced even further to respectively 38% and 17% of the baseline values, showing that the monoamine release was indeed inhibited by TTX.We have examined the long-term effects of chronic FLX treatment on extracellular monoamine concentrations in the mPFC of adult- and adolescent-treated rats. After a washout period of one week, basal extracellular levels of 5-HT, DA and NA did not differ between groups. Acute FLX caused an increase in both 5-HT and DA and NA release in all animals. The effects of prior FLX treatment on the 5-HT response to an acute challenge were clearly visible in both age groups, in that FLX-treated rats showed a twofold higher increase than saline-treated animals. A similar effect was seen on the DA response. Age-at-treatment did not influence these results.The increase in extracellular 5-HT levels produced by acute SERT blockade was significantly enhanced one week following chronic treatment. This study is the first microdialysis study looking at FLX effects to use such a long washout period. Previous studies have investigated the effects of chronic FLX treatment on 5-HT system responsiveness with shorter wash-out periods, ranging from 1–3 days. Rutter et al. (1994) and Invernizzi et al. (1996) showed that chronically treated animals have higher basal 5-HT levels 24 h after the last dosage while the latter authors reported that this effect is absent 96 h post treatment. The increases in basal levels within 24 hours after the last dosage are most probably due to reuptake blockade by the residuals of active NFLX in the brain, which can remain present up until 48 h after the last administration all treated animals, irrespective of age-at-treatment, both before and after the acute challenge. In conclusion, there were no apparent age-by-treatment effects of chronic FLX on basal extracellular 5-HT concentration and on the FLX-induced increase. Still, we cannot rule out that there are age-related differences in more down-stream processes such as post-synaptic receptor sensitivity We expected to find different effects of chronic treatment in adolescent-treated animals compared to adult-treated animals. However, such differences were not observed. As previously mentioned, a recent phMRI study by our group As hypothesized, a clear effect of the acute FLX challenge was observed on the release of extracellular DA and NA, while no effect of chronic FLX treatment on their basal levels was seen. Unpredicted however, was the small but statistically significant effect of the preceding chronic FLX treatment on DA release after the acute challenge in both age groups, just as was seen with 5-HT. Previous studies have shown that FLX can indeed acutely affect DA and NA release in the prefrontal cortex, presumably via the 5-HT2c receptor and/or post-synaptic 5-HT1A receptor So far, there is only a small number of studies that has looked into the ongoing effects of chronic FLX treatment on neurotransmitter responsiveness after its cessation. Our results show that the 5-HT system remains sensitive to external changes quite long after complete washout of the drug. While it is known from stimulant drugs that repeated administration may lead to long-lasting sensitization of the DA system A possible limitation of the present study is that we only studied the effects in one brain area, the mPFC. We should be aware that region-specific differences in 5-HT function and in response to SSRIs exist and that our findings might not be directly translatable to other brain regions, such as the raphe nuclei or the dorsal hippocampus, which is one of the other major terminal regions of 5-HT projections from the raphe Our findings indicate ongoing effects of FLX treatment on 5-HT regulation one week after drug discontinuation. After the washout period, basal extracellular 5-HT levels are normalized, indicating that the direct effects of chronic FLX on 5-HT concentrations in the rat mPFC are gone. However, when challenging the 5-HT system, we observed a hyper-responsive 5-HT system in adult- as well as adolescent-treated animals and to a lesser extent the same effect was seen on the DA system. It thus seems that both pre- and postsynaptic feedback mechanisms that should maintain stable monoamine levels are not yet completely restored one week after treatment discontinuation. These findings are of relevance, since they give us more insight in the working mechanisms of the 5-HT system and enhance our understanding of the effects of SSRIs thereupon, and might, on the long-term, have implications for clinical practice. Moreover, we can conclude that age-related difference in extracellular concentrations of 5-HT or in 5-HT responsiveness are not likely an explanation for the observations we made earlier with phMRI. Future studies focusing on more down-stream (post-synaptic) processes of 5-HT transmission, or even on other neurotransmitter systems, or studies using an even longer washout period to ensure complete restoration of the feedback mechanisms should establish if there are age-dependent changes in these specific processes.


Cardiac phase-resolved Blood-Oxygen-Level-Dependent (CP-BOLD) MRI can detect myocardial ischemia at rest without contrast and stress agents. At rest, BOLD myocardial signal intensity varies with cardiac phase: in healthy conditions it is maximal in systole and minimal in diastole, but in disease this pattern is not evident. These changes are not readily visible and post-processing is necessary. Previous methods used segmental analysis and only two images to identify ischemic segments. In this study we demonstrate that it is possible to use unsupervised learning methods to identify ischemia with higher accuracy.Flow and motion compensated 2D short-axis CP-BOLD were acquired at 1.5T along the mid ventricle in 10 canines at baseline and under severe LAD stenosis. Scan parameters were: resolution=1.2x1.2x8mm3, flip-angle=70° and TR/TE =6.2/3.1ms. For each stack, end-systolic (ES) and end-diastolic (ED) images were identified, myocardial borders were traced  and segmented in 24 radially consecutive segments, obtaining for each segment a 1D CP-resolved time series. Then time series were arranged column-wise in a matrix Y. The approach relied on the fact that remote to ischemia a cyclic pattern exists , while in ischemic territories this pattern is not the same. The divergence from this pattern is used to calculate an ischemia likelihood value (ILV). In more details, given Y, a dictionary D and a sparse representation matrix X, such that Y≈DX, are found, the dictionary D consisting of a circulant part accounting for cyclic shifts of the common pattern and a generic one. The approach iterates by finding first D, then X, and then a binary ischemia vector L, by means of the 1-class Support-Vector-Machine algorithm, which establishes if a time series is ischemic (L=1) or not (L=0) by also looking at how good it is represented by the dictionary. At each iteration time series with L=1 are excluded when updating D; the process is repeated till convergence. Finally, ILV, between 0 and 1, is calculated as a function of the support vectors and is used for visualization. Due to lack of gold standard validation of ischemia at rest, accuracy of the proposed and the Systolic to Diastolic Ratio (SDR) approach were compared using synthetic data obtained by a state-of-the-art method.In an example from a canine the proposed approach identifies correctly the ischemic territory Fig. . As seenAlthough further experiments are necessary to validate this approach, when combined with appropriate visualization and quantification methods, CP-BOLD can open the road to repeatable, truly non-invasive diagnosis of ischemic heart disease, primarily in detecting and triaging patients experiencing acute coronary syndromes and particularly in cases of NSTEMI.US National Institutes of Health, National Heart Lung and Blood Institute, (grant no: 2R01HL091989-05)Table The ratio of ischemic to total time series is known by design and fixed at 33% . Accuracy reflects how many time series were assigned a correct status (ischemia or remote) comparing the result of each approach with the ground truth assignment. The number of time series (100 or 150) was varied and several synthetic datasets (200) were generated. The average ± standard deviation over 200 experiments were reported above. Locations of end-systole and end-diastole were fixed. SDR was defined as the ratio of average segmental intensity obtained from the images at systole and diastole. A segment is ischemic if SDR <1. Statistical test (two tailed paired t-test) shows significant difference in performance among the two approaches.


Drosophila melanogaster via the modENCODE project. We classified 96 core promoters into four groups based on the presence or absence of the TATA box or DPE, calculated the histone modification ratio at the core promoter region, and transcribed region for each core promoter. We found that the histone modifications in TATA-less groups were static during development and that the core promoters could be clearly divided into three types: i) core promoters with continuous active marks (H3K4me3 and H3K27ac), ii) core promoters with a continuous inactive mark (H3K27me3) and occasional active marks, and iii) core promoters with occasional histone modifications. Linear regression analysis and non-linear regression by random forest showed that the TATA-containing groups included core promoters without histone modifications, for which the measured RNA expression values were not predictable accurately from the histone modification status. DPE-containing groups had a higher relative frequency of H3K27me3 in both the core promoter region and transcribed region. In summary, our analysis showed that there was a systematic link between the existence of the CPEs and the dynamics, frequency and influence on transcriptional activity of histone modifications.Four different histones  constitute a histone octamer, around which DNA wraps to form histone-DNA complexes called nucleosomes. Amino acid residues in each histone are occasionally modified, resulting in several biological effects, including differential regulation of transcription. Core promoters that encompass the transcription start site have well-conserved DNA motifs, including the initiator (Inr), TATA box, and DPE, which are collectively called the core promoter elements (CPEs). In this study, we systematically studied the associations between the CPEs and histone modifications by integrating the Drosophila Core Promoter Database and time-series ChIP-seq data for histone modifications  during development in  Drosophila melanogaster. For this purpose, researchers have collected comprehensive data, including RNA-seq for transcripts and ChIP-seq for modified histones, under different experimental conditions.As massively parallel DNA sequencing by next-generation sequencing (NGS) is gaining popularity, biologists have gained better access to genome-wide analysis using NGS-based techniques such as chromatin immunoprecipitation (ChIP)-seq (the combination of ChIP and NGS to determine the locus at which a protein of interest is bound) or RNA-seq (quantitative detection of transcripts by NGS). Moreover, the enhancement of biological databases has been made possible through the efforts of scientists involved in large-scale projects such as the modENCODE project . This prHistones are proteins that function to compact DNA. Histone octamers are formed by two of each of four different histones ; DNA wraps around the histone octamer to form a histone-DNA complex called a nucleosome [D. melanogaster [D. melanogaster [D. melanogaster [D. melanogaster [Many of the recent studies have focused on the core promoter region of RNA polymerase II, which contains the transcription start site (TSS), as some histone modifications, such as H3K4me3, are known to occur around TSSs . In partnogaster ) encompanogaster ), whose nogaster , is the nogaster . The DPEnogaster ) functionogaster  and is pnogaster . This elnogaster . Studiesnogaster .D. melanogaster [The purpose of this study was to provide insights into the transcription system regulated by the histone modification status and the combination of CPEs. We hypothesized that basal transcription machinery is dependent on histone modification and the CPEs may function cooperatively; therefore, the patterns of histone modifications may be affected by the specific combination of CPEs. For example, Negre et al. produced a large-scale dataset that detected the status of histone modifications such as H3K4me3, H3K27ac, and H3K27me3 by ChIP-seq to study the dynamics of histone modifications and determined the quantity of each transcript by RNA-seq at several developmental stages from the early embryo stage to the adult stage in CP; T; p-values = 1.49387e-71 and DPE: empirical p-values = 4.181458e-66): i) continuously high HMRs in active marks (H3K4me3 and H3K27ac) and continuously low HMRs in inactive marks (H3K27me3), ii) continuously high HMRs in inactive marks (H3K27me3) and few active marks (H3K4me3), and iii) low HMRs , only TATA , only DPE , and both TATA and DPE . For each core promoter region, we computed the histone modification ratio (HMR) as the ratio of the histone-enriched region (determined by the ChIP-seq dataset in GSE15292) to the entire length of the core promoter HMRCP; . Additiolow HMRs . The TATlow HMRs .Correlatlow HMRs . These rp-value  and its r = 0.593, p-value < 2.2e-16Inr: r = 0.613, p-value < 2.2e-16DPE: r = 0.465, p-value < 2.2e-16TATA: r = 0.357, p-value = 2.16e-07TATA-DPE: T and K4me3CP were positively selected. In DPE-less groups, K27me3CP was negatively selected. Interestingly, TATA-less and TATA-containing groups differed in terms of the predicted log(FPKM) versus the measured log(FPKM)  . In the r = 0.533, p-value = 2.03e-14TATAp: r = 0.245, p-value = 0.000819TATA-DPEp: T and K4me3CP were positively selected. In both groups, K27me3CP was negatively selected. The correlation coefficient of TATA was improved from 0.465 to 0.533 by removing TATAn, while this improvement was not observed in the TATA-DPE group (r changed from 0.357 to 0.245).To examine the importance of each histone modification for transcription, we determined the relative importance of each explanatory variable in the regression equation using LMG [T was the most influential histone modification on RNA expression levels in all groups except for DPE; K4me3CP was the second most influential modification. Interestingly, K27me3CP was influential in TATAp and TATA-DPEp, regardless of their negative coefficients  for the presence of TATA and the pattern of scatter plot. The measured and predicted values however might have some non-linear correlations, which might result in the unique pattern in T is higher than that of H3K4me3T in all groups except for DPE. Moreover, the importance of H3K27me3CP in TATA-containing groups  was higher than that in TATA-less groups .We compared the importance of each explanatory variable in random forest (mean decrease in node impurity in CP and K27acT) influenced transcription according to linear regression analysis.We created a heatmap of the ratio of core promoters with HMRs of more than 0.5 to compare the frequencies of histone modifications . As showT and K4me3CP, In summary, TATA-less core promoters showed small dynamic changes and could be divided into three types: i) continuous with active marks, ii) continuous with inactive marks, and iii) void state and transcriptionally inactive. Since the inactive mark rarely occurred in the first type, the RNA expression values could be predicted by the status of the active marks alone  and phosphorylated p53 and subsequent recruitment of histone deacetylase 1 (HDAC1) mediate the repression of insulin-like growth factor 1 (IGF1R) expression caused by oxidative stress . Direct The characteristic distributions of histone modifications have been studied intensively, and it is reported that H3K4me3 is enriched at sites of transcription initiation  and thatD. melanogaster are available to compare with the data we used for this analysis. Considering the result of regression analysis in Figs One of our most exciting findings was the void state in the TATA-containing groups, whose RNA expression values seemed independent of histone modification status. There are three possible explanations for this phenomenon. First, the TATAn and TATA-DPEn groups were not occupied with histones and exhibited TATA-specific transcriptional regulation. Second, these groups may have unmodified histones and exhibit TATA-specific transcriptional regulation. Third, these groups may be in the void state and transcriptionally inactive, despite changes in RNA stability. The first explanation is partly supported by a report showing that the TBP, which is included in basic RNA polymerase II transcription machinery, requires a nucleosome-free region to bind to the core promoter region . We perfWe found that the DPE and TATA-DPEp groups had a higher relative frequency of H3K27me3. Although it is unclear whether this tendency occurs only during development, these data implied that the presence or absence of the DPE sequence affected the histone modification patterns. Models of the relationships between histone modifications and CPEs are shown in All ChIP-seq GSE16013) and RNA-seq (GSE18068) data are included in GSE15292 , obtaine6013 and http://labs.biology.ucsd.edu/Kadonaga/DCPD.html) [We obtained DNA sequences of promoters that had only the Inr sequence and no TATA box or DPE sequence , promoters that had a TATA box but no DPE sequence , promoters that had a DPE sequence but no TATA box , and promoters that had both a TATA box and DPE sequence  from the Drosophila Core Promoter Database  . Genes thttp://hannonlab.cshl.edu/fastx_toolkit/index.html). Sequenced reads were mapped using bowtie-1.0.0 [D. melanogaster). To calculate the histone modification ratio at the core promoter region, we created a BED format file for each CPE group by submitting the DNA sequences obtained from DCPD to BLAST in FlyBase [We downloaded the raw ChIP-seq data in FASTQ format and checked the sequence quality using a FASTX-toolkit (ie-1.0.0 . We builie-1.0.0  and convie-1.0.0 . For peaie-1.0.0  with an  FlyBase . For the FlyBase  in BED fD. melanogaster Ensembl BDGP5.25) from the TopHat website (http://ccb.jhu.edu/software/tophat/igenomes.shtml) and used this information to run TopHat. To calculate the FPKM for each transcript, Cufflinks-2.1.1 [After the sequence quality check, carried out as with the ChIP-seq data, the sequenced reads were mapped using TopHat-2.0.8b , 39 withks-2.1.1 , 40–42 whttp://cran.r-project.org/web/packages/pheatmap/index.html) was used to create the heatmaps.The pheatmap package in R  cluster centers were then randomly generated to compute the distance in the same way as was performed for the correct distance. This random generation (and distance computation) was performed one million times to check whether the correct distance occurred significantly, yielding the empirical http://cran.r-project.org/web/packages/leaps/index.html) was used for an exhaustive search for the best combination of variables. This returned separate best models of all sizes, and we selected one model whose explanatory variables all significantly affected the objective variance (p < 0.05 using the hypergeometric test). In the TATA-DPE group, we selected the model in which one of the explanatory variables (K27me3TSS) was not significant , because the relative importance of this variance was reasonably high, as shown in http://cran.r-project.org/web/packages/bootstrap/index.html). To obtain the regression equations for the TATAp group, we removed observations in the TATA group for which the predicted log(FPKM) values ranged from 0.7 to 1. The removed observations were treated as in the TATAn group. For the TATA-DPEp group, we removed observations whose predicted log(FPKM) values ranged from 0 to 1. The relative importance of each explanatory variable was calculated by the LMG method, using the relaimpo package in R [Linear regression analysis was carried out as previously described , 44. We age in R . The matThe randomForest package in R  was usedS1 FigThe y-axis represents histone modification ratios, and the x-axis represents the CPE groups. Their orders reflect the results of hierarchical clustering by the pheatmap package in R. The correlation coefficients between RNA expression values and histone modification ratios were visualized by heatmap.(PDF)Click here for additional data file.S2 Fig(A) Scatterplot showing correlations among histone modifications. (B) A scatterplot was used to check the homogeneity of variance. (C) The homogeneity of variance was checked using a scale different from that used in (B). (D) A normal Q-Q plot was used to check the normal distribution of errors. (E) Cook’s distance was used to identify influential observations.(PDF)Click here for additional data file.S3 Fig(A) A scatterplot was used to show correlations among histone modifications. (B) A scatterplot was used to check the homogeneity of variance. (C) The homogeneity of variance was checked using a scale different from that in (B). (D) A normal Q-Q plot was used to check the normal distribution of errors. (E) Cook’s distance was used to identify influential observations.(PDF)Click here for additional data file.S4 Fig(A) Scatterplot showing the correlation among histone modifications. (B) A scatterplot was used to check the homogeneity of variance. (C) The homogeneity of variance was checked using a scale different from that used in (B). (D) A normal Q-Q plot was used to check the normal distribution of errors. (E) Cook’s distance was used to identify influential observations.(PDF)Click here for additional data file.S5 Fig(A) Scatterplot showing correlations among histone modifications. (B) A scatterplot was used to check the homogeneity of variance. (C) The homogeneity of variance was checked using a scale different from that used in (B). (D) A normal Q-Q plot was used to check the normal distribution of errors. (E) Cook’s distance was used to identify influential observations.(PDF)Click here for additional data file.S6 FigThe y-axis represents the histone modification ratio, and the x-axis represents histone modifications. (A) Boxplot of histone modification ratios in the TATAp group (n = 189). (B) Boxplot of histone modification ratios in the TATA-DPEp group (n = 51). (C) Boxplot of histone modification ratios in the TATAn group (n = 174). (C) Boxplot of histone modification ratios in the TATA-DPEn group (n = 103).(PDF)Click here for additional data file.S7 FigGO analysis was performed using DAVID Bioinformatics Resources 6.7. The enriched functional categories (SP_PIR_KEYWORDS) with FDRs of less than 1% were selected.(PDF)Click here for additional data file.S8 FigThe k-means clustering was performed with 1000 iteration to obtain five clusters for each CPE group. The distribution of the histone modification ratios in each cluster was visualized by boxplot. The y-axis represents the histone modification ratio, and the x-axis represents the histone modification. The clusters were sorted from top to bottom according to their numbers of core promoters. Bold lines represent clusters in which the medians of histone modifications were equal to zero.(PDF)Click here for additional data file.


S)-naproxen anhydrate–hydrate system.Relationships between the crystal structures of two polymorphs of sodium naproxen dihydrate and its monohydrate and anhydrate phases provide a basis to rationalize the observed transformation pathways in the sodium ( S)-naproxen. The structure of DH-I is determined from twinned single crystals obtained by solution crystallization. DH-II is obtained by solid-state routes, and its structure is derived using powder X-ray diffraction, solid-state 13C and 23Na MAS NMR, and molecular modelling. The validity of both structures is supported by dispersion-corrected density functional theory (DFT-D) calculations. The structures of DH-I and DH-II, and in particular their relationships to the monohydrate (MH) and anhydrate (AH) structures, provide a basis to rationalize the observed transformation pathways in the sodium (S)-naproxen anhydrate–hydrate system. All structures contain Na+/carboxylate/H2O sections, alternating with sections containing the naproxen molecules. The structure of DH-I is essentially identical to MH in the naproxen region, containing face-to-face arrangements of the naphthalene rings, whereas the structure of DH-II is comparable to AH in the naproxen region, containing edge-to-face arrangements of the naphthalene rings. This structural similarity permits topotactic transformation between AH and DH-II, and between MH and DH-I, but requires re-organization of the naproxen molecules for transformation between any other pair of structures. The topotactic pathways dominate at room temperature or below, while the non-topotactic pathways become active at higher temperatures. Thermochemical data for the dehydration processes are rationalized in the light of this new structural information.Crystal structures are presented for two dihydrate polymorphs (DH-I and DH-II) of the non-steroidal anti-inflammatory drug sodium ( Our interest lies principally with pharmaceutical compounds, for which robust physicochemical understanding is paramount (Connelly S)-naproxen  sodium -naproxen anhydrate  was obtained from Divi’s Laboratories Ltd, India. Methods for preparation of the various bulk phases have been described previously  at 60°C. The solution was filtered to remove any undissolved residues of AH then allowed to cool to room temperature. The first single crystals to appear were transferred rapidly from the mother liquor to perfluoropolyether oil, then into the N2 cryostream (at 150 K) on the diffractometer. Several crystals from different batches were examined, with comparable results.Sodium (2.2.Kα radiation (λ = 0.7107 Å). Data were collected at 150 K under an N2 cryostream in an effort to minimize dehydration during the data collection. Structure solution and refinement were carried out using SHELXL . Data were collected in either flat-plate reflection or transmission capillary modes. Preliminary data analysis was carried out using HighScorePlus  calculations, using the 3.3.1.et al., 2004et al., 1990+/carboxylate/(H2O) sections alternating with sections containing the naproxen molecules . In AH –Na+ and Na+–[O–C–O]–Na+. The latter define polymeric ribbons along the crystallographic b axis. The Na+/carboxylate sections are locally centrosymmetric. If the naproxen molecules are deleted so that only the Na+ ions and carboxyl groups remain, the structure can be described in space group P21/c with one crystallographically distinct Na+ ion and one carboxylate group. Reduction of the symmetry to P21 arises due to the naproxen molecules, which adopt an edge-to-face type arrangement 2–Na+ units, in which the μ-O linkages are provided by the carboxyl groups. The water molecules project to either side, linking the ribbons along the a axis through O—H⋯O hydrogen bonds. The Na+/carboxylate/H2O sections contain local inversion centres that intersect the crystallographic 21 screw axes so that the space group approximates P21/m. Again, the inversion symmetry is broken by the naproxen molecules, which in this case adopt a face-to-face type arrangement, with all naphthalene ring planes parallel. The Na+ coordination geometry resembles square-based pyramidal, with Na+ lying out of the approximate square plane. In contrast to AH, the ring planes of the naproxen molecules lie approximately eclipsed with the C11—C13 bond when viewed in projection along the C10—C11 bond  is highly sensitive towards the hydration state. The parameters in Table 2supporting information) show that the quadrupolar coupling constant CQ is 2.3 MHz or greater for AH and both DH forms, while it is close to 1.0 MHz for MH. The presence of two 23Na sites in AH, as indicated by the crystal structure  mainly indicate a distribution in quadrupolar tensor parameters rather than a distribution of chemical shifts.es Fig. 5 demonstr13C CP/MAS NMR spectra , on account of limited observable data and the low symmetry of the structure, and it was necessary to apply restraints to all bond distances and angles in order to maintain a reasonable geometry. The problems with the single-crystal analysis most likely also reflect some degree of dehydration during the transfer of the crystals from the mother liquor to the N2 cryostream and/or in the course of the data collection. The validity of the established structure is supported by DFT-D minimization, which results in an r.m.s. Cartesian displacement of 0.14 Å for the non-H atoms, comparable to that obtained for minimization of AH and MH. Comparison to the PXRD pattern of the bulk sample by Rietveld refinement also provides a satisfactory fit (see supporting information).Numerous solution-grown crystals of DH-I were examined, and all displayed indications of twinning/disorder (discussed further below). The structure was eventually obtained after data integration using a single component in one crystal, although subsequent structure refinement was problematic. In particular, the data:parameter ratio is low 2–Na+ units. The linking units are geometrically similar to those in AH, except that the μ-O bridges in DH-I are formed by water molecules  appear ordered for 0kl, 1kletc., indicative of regular two-dimensional layers in the structure parallel to the (100) planes. The disorder is evident in the diffraction pattern as reflections split along a*. The probable origin of this can be viewed as a result of the pseudosymmetry. Combination of the local 21 operator that relates the naproxen molecules (+/H2O sections generates a mirror operator (+/H2O section to reverse the orientation of the square-shaped Na+–(μ-OH2)2–Na+ units relative to the b axis . However, the γ angle deviates sufficiently from 90° for the twinning to be apparent as split peaks in the diffraction pattern, as described. Attempts at two-component integration of the single-crystal data were not successful, apparently due to the close overlap of the diffraction peaks. Post-analysis of the data integrated as a single component using TWINROTMAT in PLATON . The resulting enlarged unit cell could be transformed to the AH-type cell obtained from Pawley fitting by application of the matrix [1 0 −½ / 0 1 0 / 0 0 1]. The result corresponds to a supercell of the DH-I structure described in the AH-type cell setting .The applied preparation route for DH-II, by direct hydration of solid AH, prohibits the formation of diffraction-quality single crystals, and the DH-II structure was therefore considered using a combined PXRD/modelling approach. The ra Fig. 5 show thara Fig. 5 to proviis Fig. 9a. The rng Fig. 9a.b in order for the naproxen molecules to adopt positions similar to those in AH . Crucially, this shift is permitted by the fact that the water molecules along the coordination polymers are distributed at intervals of approximately ½b, as shown in Fig. 8+/carboxylate/H2O region overlays essentially exactly the comparable region in the dihydrate of the sodium salt of ibuprofen . As a final step to model the DH-II structure, the naphthalene rings of both naproxen molecules in the shifted polymeric ribbon were rotated around the C5—C11 bond to emulate the ring positions in AH. The final model comprises Na+/carboxylate/H2O regions similar to those in Ibu-DH, with naproxen regions similar to those in AH. The broader lines in the 13C CP/MAS NMR spectra for DH-II and lack of singularities in the 23Na MAS NMR lineshapes . The line broadening seen in the 13C NMR, and the multiple resonances for C11 and C13, can be attributed to the presence of the two alternative molecular conformations .(i) For DH-I, topotactic transformation to MH operates initially, while non-topotactic transformation to AH is activated only above a threshold temperature. In the TGA curve at 1°C minvia MH is activated only above a threshold temperature. At 1°C min−1, the DH-II → AH transformation is almost complete before the non-topotactic threshold temperature is reached, so the plateau for MH is small and it appears at a lower weight % because the MH produced from the DH-II that remains at that time comprises only a small fraction of the total sample. At higher heating rates, the non-topotactic pathway is activated while there is still a significant amount of DH-II present, so the plateau for MH appears more pronounced and closer to the expected 93 wt %.(ii) For DH-II, topotactic transformation to AH operates initially, while the non-topotactic pathway et al.  Malaj  al. 2009 have preWith the established structural information, we interpret the TGA data as follows:via DH-I. Conceptually, this depends on the rate for rearrangement of the naproxen molecules from edge-on (in DH-II) to parallel (in DH-I), compared with the rate at which the water stoichiometry changes from dihydrate to monohydrate. If the naproxen rearrangement occurs before the water stoichiometry changes, the transformation proceeds via DH-I. If the stoichiometry changes simultaneously with the naproxen rearrangement, the transformation proceeds directly from DH-II to MH. The DFT-D minimized structures of MH and DH-I have essentially identical unit-cell volumes (see supporting information), which indicates that the packing arrangement is governed by the naproxen molecules and that the Na+/H2O region of either MH or DH-I can be accommodated within the same framework of naproxen molecules. The implication is that the hydrate stoichiometry for a crystalline material having the MH/DH-I structure could be continuously variable between MH and DH. In this case, the distinction between direct DH-II → MH or sequential DH-II → DH-I → MH transformation has little practical meaning, and the process is better represented as DH-II → [DH-I ↔ MH]. There is also a possibility that the water stoichiometry could change from DH to MH before any naproxen rearrangement occurs. This would produce a polymorph of MH having the edge-on naproxen structure (i.e. ‘MH-II’). We examined this possibility using PXRD and ss-NMR under various in situ dehydration conditions, but we did not find any evidence for such a phase. Likewise, we did not find any evidence for an AH phase having a parallel naproxen arrangement.For dehydration of DH-II to AH, it is interesting to consider the sequential DH-II → MH → AH pathway, which involves two non-topotactic steps and operates above room temperature. For the DH-II → MH step, we might speculate on whether the transformation occurs directly, or sequentially 4.S-NMR approach becomes crucially important. Although the experimental and computational effort required to apply these techniques is significantly greater than that for a contemporary single-crystal X-ray analysis, the rewards for understanding systems of this type are significant.The structural data provided here for the sodium naproxen anhydrate–hydrate system enable us to rationalize the complex transformation pathways that have previously been observed. The key is to establish the topotactic and non-topotactic nature of the various transformations. For this exercise to be effective, it is clearly important to have structural information for all involved solid phases. For DH-II in particular, the required solid-state preparation route hinders growth of suitable single crystals, and the combined modelling/PXRD/10.1107/S2052252514015450/ed5002sup1.cifCrystal structure: contains datablock(s) DH1, DH2. DOI: 10.1107/S2052252514015450/ed5002DH1sup3.hklStructure factors: contains datablock(s) DH1. DOI: 10.1107/S2052252514015450/ed5002DH2sup4.rtvRietveld powder data: contains datablock(s) DH2. DOI: 10.1107/S2052252514015450/ed5002sup2.txtCIF with DFT-D minimised structures for AH, MH, DH-I, DH-II. DOI: 10.1107/S2052252514015450/ed5002sup5.pdfExperimental details and additional figures for SS-NMR; reconstructed precession images for DH-I; displacement ellipsoid plot for DH-I; details of Rietveld refinements; comparison of DH-II and sodium ibuprofen dihydrate. DOI: 1019881, 1019882CCDC references:


Iron dextran also increased mRNA levels of apoB-100, MTP, and L-FABP indicating alterations in lipid secretion. Carbonyl protein and TBARS were consistently higher in the liver of the iron-treated rats. Moreover, a significant positive correlation was found between oxidative stress products, lfabp expression, and iron stores. In addition, a negative correlation was found between pparα expression, TBARS, carbonyl protein, and iron stores. In conclusion, our results suggest that the increase observed in the transport of lipids in the bloodstream and the decreased fatty acid oxidation in rats, which was promoted by iron dextran, might be attributed to increased oxidative stress.The objective of this study was to investigate the effects of iron dextran on lipid metabolism and to determine the involvement of oxidative stress. Fischer rats were divided into two groups: the standard group (S), which was fed the AIN-93M diet, and the standard plus iron group (SI), which was fed the same diet but also received iron dextran injections. Serum cholesterol and triacylglycerol levels were higher in the SI group than in the S group. Iron dextran was associated with decreased mRNA levels of  This ef srebp-2 [ srebp-2 can regulate cholesterol homeostasis via its ability to bind and activate the promoters of genes encoding LDL receptor (LDL-R) and HMG-CoA reductase (HMG CoA-R).In the liver, a complex network of nuclear receptors coordinately regulates the expression of enzymes involved in different steps of lipid metabolism, from fatty acid (FA) oxidation and uptake to triacylglycerol (TAG) synthesis, accumulation, and/or secretion . Sterol  srebp-2 , which p srebp-2 . srebp-2α (pparα) is a transcription factor involved in liver FA degradation. It is expressed in the liver where it accelerates the transcription of proteins and enzymes for FA catabolism. It plays an important role in the metabolic homeostasis of FAs through the regulation of target genes encoding enzymes for FA oxidation and transport, such as carnitine palmitoyltransferase 1 (CPT1), which controls mitochondrial β-oxidation. A growing body of evidence supports a role for pparα in the development of liver disease, and disabling the pparα gene is known to increase hepatic triglyceride accumulation, especially under fasting conditions [ pparα [Peroxisome proliferator-activated receptor nditions , 12. In  [ pparα .Triglyceride release as very low-density lipoprotein (VLDL) requires the coordinated functions of apolipoprotein B-100 (apoB-100) and microsomal triglyceride transfer protein (MTP) . ApoB-10To further investigate the hyperlipidemic effect of iron dextran and the possible underlying mechanism, we first focused on its effects on serum cholesterol and triacylglycerol levels. To identify the mechanisms of the hyperlipidemic effects at the molecular level, we determined mRNA expression of genes involved in lipid oxidation and triglyceride and cholesterol release from the liver and the association with enhanced oxidative stress.n = 14), weighing approximately 180 g, were obtained from the School of Nutrition, Federal University of Ouro Preto, MG, Brazil. Animals were individually housed in wire-bottomed metabolic cages and kept in a room with controlled conditions and food and water provided ad libitum. The standard group (S) was fed a standard diet (AIN-93M). The standard plus iron group (SI) was fed the standard diet and was given intraperitoneal injections of 100 g/L iron dextran . For all experiments, injections were given at a total dose of 40 mg, divided into 10 mg/d dose, according the protocol by Miron et al. [Eight-week-old male Fischer rats  level was determined from the supernatant using Labtest kit number 13. The level of other cholesterol fractions was calculated as the difference between the levels of total and HDL cholesterol. In addition, Labtest kits 53, 42, and 84 were used to measure the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glucose concentration, respectively.The liver concentration of nonheme iron was analyzed after digestion in 2 mL of acid solution (3 mol/L HCl and 10% trichloroacetic acid) for 20 h at 65°C. Digested samples were mixed with chromogen reagent containing orthophenanthroline . Serum tLiver samples from all rats were homogenized in 0.1 M Tris-HCl buffer (pH 7.4). Peroxy lipid content was measured according to Buege and Aust  and was μm were obtained by sectioning embedded fragments on a rotary microtome. Sections were mounted on glass slides previously cleaned and degreased. Slides were stained with hematoxylin and eosin for the visualization of histological changes and with Perls' technique for determination of tissue iron pools.Liver fragments not exceeding 4 mm in diameter were fixed in 10% formaldehyde solution and then dehydrated, diaphanized, and embedded in paraffin. Paraffin sections of approximately 4 μL reaction using MultiScribe (50 U/μL) Reverse Transcriptase and Oligo d(T)16 primers . Gene expression was analyzed using SYBR Green PCR Master Mix (Applied Biosystems). The primers used to amplify pparα, srebp-2, HMG CoA reductase, the LDL receptor, and the reference gene 18S have been previously described by Li et al. [ Rattus norvegicus genome  and were constructed using the Primer-BLAST Program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primer sequence for MTTP (forward primer 5′-CGACGGTGACGATGATCAACT-3′ and reverse primer 5′-TGACCCGCATTTTCGACATT-3′) was also derived from the R. norvegicus genome . The primer sequence for L-FABP (forward primer 5′-CATCCAGAAAGGGAAGGACA-3′ and reverse primer 5′-CACGGACTTTATGCCTTTGAA-3′) was derived from the R. norvegicus genome . The primer sequence for cpt-1a (forward primer 5′-ATATTGGGCACAGTCCCCTG-3′ and reverse primer 5′-TGTGAAGAAACAACCCCCAGA-3′) was derived from the R. norvegicus genome .TRIzol RNA extraction was completed using the RNAgents Promega-Total RNA Isolation System  according to the manufacturer's recommendations. Total RNA concentration and purity were determined by spectrophotometric analysis at 260 and 280 nm on a NanoVue spectrophotometer . cDNA was synthesized from total RNA using GeneAmp RNA PCR . Briefly, cDNA was prepared in a 20 i et al. , Xiong ei et al. , and Reei et al. . The priPrimers were previously tested in conventional reverse transcription PCR, in which a single band of the expected amplicon size was obtained. Quantitative (q)PCR was performed using an ABI 7300 Real Time PCR Instrument (Applied Biosystems) for 40 cycles at 95°C for 15 sec and at 60°C for 60 sec. Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that a single PCR product was detected. Each sample was run in triplicate, and PCR reactions without the addition of the template were used as blanks. The results were normalized to the reference gene, and fold changes in expression were calculated using threshold cycle (Ct) values.t-test. Correlation calculations were done using the Pearson correlation coefficient. In all tests, the results were considered significant with P values ≤ 0.05.Data are expressed as the means ± standard deviation (SD). All data were analyzed using the Kolmogorov-Smirnov normality test and were normally distributed. Statistical comparisons were made using Student's P < 0.0001) in group SI relative to group S  in group SI than in group S. The same profile was observed for the concentration of cholesterol in other fractions. HDL-C levels in group S and group SI were not significantly different. Regarding triacylglycerol concentrations, again the rats in group SI showed higher levels compared with group S, and a similar profile was observed for glucose concentration. No difference between the groups was detected in aminotransferase activities.As can be observed in P < 0.0001) than those in group S. Furthermore, a 2.13-fold increase (P < 0.0001) in protein oxidation caused by iron overload was seen in group SI compared with group S.As shown in Histological analysis showed that, generally, the liver of animals in group S was normal . The anisrebp-2 was lower (P > 0.01) in group SI than in group S ( apoB100 was higher (P > 0.01) in group SI than in group S  was recently found as regulator of hepatic lipid metabolism [cpt-1a expression was markedly decreased in iron dextran, which is in agreement with previous studies by Koonen et al. [ pparα and its target genes in the liver.sporters , 12. ppanditions –40. Pharepatitis , 41. Furepatitis , and theepatitis . Iron deo et al.  showed so et al. , suggesttabolism . We founn et al.  and Lelln et al. , who obs apoB-100, which were promoted by iron dextran, suggest that an increase in the overall secretion of VLDL is caused by modifications in the packaging of this lipoprotein. In addition to the upregulatory effect on apoB-100, supplementation of iron increased the expression of MTP in this study. MTP plays an important role in VLDL assembly by mediating the transfer of hepatic lipids to nascent apoB [ MTP gene expression is enhanced in the liver of both fructose-fed hamsters and obese diabetic mice [MTP mRNA expression was significantly increased in iron overloaded rats, which may contribute to increased plasma cholesterol and triacylglycerols, as MTP inhibitors have been developed and shown to be effective lipid-lowering drugs in animal models [ApoB-100 is associated with hepatic-derived non-HDL-C and is incorporated into nascent lipoprotein particles, along with cholesterol and triglycerides . The incent apoB . It has tic mice . In the l models . Studiesl models , 50. The L-FABP mRNA was increased in comparison to the control group [L-FABP is one of the most abundant proteins in the liver, accounting for 2-3% of the total cytosolic protein pool . As suchol group . Thus, tol group  and formol group . The posol group  showed t srebp-2 stimulates hypercholesterolemia and hepatic steatosis because srebp-2 is activated in response to the reduction in hepatic cholesterol content. However, the alterations observed in srebp-2, and the genes regulated by it (HMG-CoA reductase and LDL-R) does not seem to be involved in the mechanism of action of iron promoting hypercholesterolemia. Iron-treated rats showed higher serum cholesterol levels and reduced expression of srebp-2 (and tendency of reduced expression of HMGCR and LDLR). The reduction in srebp-2 expression may indicate feedback regulation in response to high cholesterol levels.The mRNA level of the other factors involved in FA metabolism (including SREBPs) was significantly increased in the livers of mice under conditions of elevated oxidative stress . FurtherThe present study demonstrated that iron dextran increased oxidative stress, which was associated with the altered expression of genes related to lipid metabolism and therefore contributing to hyperlipidemia.


P = 0.047). bRFS in the pre-HDR PSA ≤ 0.1 ng/ml subgroup was significantly improved compared with that in the pre-HDR PSA > 0.1 ng/ml subgroup . On multivariate analysis, dose of HDR  and pre-HDR PSA level  were significant prognostic factors predicting bRFS. In high-risk prostate cancer patients treated with the trimodality therapy, the dose of HDR and pre-HDR PSA were significant prognostic factors. The pre-HDR PSA ≤ 0.1 subgroup had significantly improved bRFS. Further studies are needed to confirm the relevance of pre-HDR PSA in trimodality therapy.Trimodality therapy consisting of high dose rate (HDR) brachytherapy combined with external beam radiation therapy (EBRT), neoadjuvant hormonal therapy (NHT) and adjuvant hormonal therapy (AHT) has been used to treat localized high-risk prostate cancer. In this study, an analysis of patients receiving the trimodality therapy was performed to identify prognostic factors of biochemical relapse-free survival (bRFS). Between May 2005 and November 2008, 123 high-risk prostate cancer patients (D'Amico classification) were treated with NHT prior to HDR brachytherapy combined with hypofractionated EBRT. Among these patients, 121 had completed AHT. The patients were assigned by time to be treated with a low-dose or high-dose arm of HDR brachytherapy with subsequent hypofractionated 3D conformal radiation therapy (3D-CRT). Multivariate analysis was used to determine prognostic factors for bRFS. With a median follow-up of 60 months, the 5-year bRFS for all patients was 84.3% (high-dose arm, 92.9%; low-dose arm, 72.4%,  There are many options for radical radiotherapy for localized prostate cancer. In addition to conventional external beam radiation therapy (EBRT), such as 3D conformal radiotherapy (3D-CRT) and intensity modulated radiotherapy (IMRT), brachytherapy, such as low-dose rate (LDR) or high-dose rate (HDR) brachytherapy, is performed. While efficacy has been reported for all modalities in low-to intermediate-risk prostate cancer, it has been reported that the efficacy of monotherapy with radiation in high-risk prostate cancer is limited .There have been many reports that dose escalation is correlated with PSA-free survival in prostate cancer, and it is known that the contribution of dose escalation is more marked in higher risk prostate cancer . In currBased on the results of long-term adjuvant hormonal therapy (AHT) as multimodality treatment for high-risk prostate cancer reported by the EORTC , long-teA total of 123 high-risk localized prostate cancer patients who received NHT, HDR brachytherapy, 3D-CRT and AHT between May 2005 and November 2008 were included in the study. Follow-up was commenced at the initiation of HDR, and follow-up of the last patient with trimodality therapy started in November 2008. The D'Amico classification system was used to classify the risk. The median age of patients was 69 years (range: 59–82), and the median initial PSA was 23.5 ng/ml (range: 3.9–365). The Gleason score (GS) was 6, 7, 8, 9 and 10 in 10, 32, 42, 32 and 7 patients, respectively, with a median score of 8. The median follow-up was 60 months (range: 26–88), and the median duration of NHT was 8 months (range: 1–29). The median duration of AHT was 24 months (range: 3–24), and 98% of patients received AHT for 24 months , which was the gross target volume (GTV = prostate) with a 4-mm margin around the prostate (except 2-mm margin only on the rectum) was treated at the prescribed dose. The total dose of HDR brachytherapy consisted of two arms: a low-dose arm in the early study period and a high-dose arm in the late study period. In the low-dose arm, the above-mentioned PTV was treated twice in a day with a first dose of 5 Gy and a second dose of 6 Gy . We administered 5 Gy, 6 Gy to the PTV. The maximum urethral dose was kept to 150% of the prescription dose. In the high-dose arm, a total dose of 18 Gy was administered once daily for 2 d (9 Gy × 2 fx). In a 3D-CRT setting, the PTV—defined as the GTV  with a 5-mm margin—plus an additional 5-mm PTV margin was treated at the prescribed dose. Combined 3D-CRT was administered at total dose of 45 Gy (3 Gy/fx) for the low-dose arm and 40 Gy for the high-dose arm (2.5 Gy/fx). PSA failure following trimodality therapy was defined according to the Phoenix/ASTRO definition. We compared the two arms in terms of population by Mann-Whitney's U tests. bRFS's were compared with the Kaplan–Meyer method and log rank statistics. Multivariate analysis was performed to analyze prognostic factors in this study using SPSS This study was conducted in accordance with the Helsinki Declaration and the intramural ethics committee. Written informed consent was obtained prior to radiotherapy.P = 0.047) . The treatment outcome in the patients stratified by the duration of NHT into three arms  is shown in Fig. P = 0.2).The median duration of follow-up was 60 months, and the 5-year bRFS was 84.3% in the entire population. The 5-year bRFS was 92.9% in the high-dose arm, and 72.4% in the low-dose arm (47) Fig. . The 5-yP = 0.034).The treatment outcome for the patients stratified by the PSA value (pre-HDR PSA) immediately before HDR brachytherapy following NHT (which greatly varied) into two arms (≤0.1 ng/ml and >0.1 ng/ml) is shown in Fig. P = 0.045, HR : 0.25, 95% CI : 0.038–0.97) and pre-HDR PSA level  were significant prognostic factors predicting bRFS    and pre-HDR PSA level (P = 0.02) were significant prognostic factors predicting bRFS in multivariate analysis  Figs  and 3. Aet al. reported that the post-NHT PSA nadir, rather than the duration of the NHT, was important in NHT [As for the duration of NHT, the treatment outcome was worse in the ‘6 months < HT ≤ 12 months’ arm than expected. The cause remains unknown, since the percentage of patients with pre-HDR PSA ≤ 0.1 ng/ml and of those who received LDR brachytherapy was not unfavorable in this arm as compared with the ‘NHT ≤ 6 months’ arm. Liudgate t in NHT .In the Canadian trial , 6, it wOur retrospective study did not demonstrate i-PSA and GS as significant prognostic factors, although pre-HDR PSA and HDR dose were significant prognostic factors. Lower value of pre-HDR PSA is supposed to imply less tumor burden , 13. It et al. reported that the tumor area was hypoxic in prostate cancer, and a tumor with low pO2 was associated with a higher risk of early PSA recurrence [et al. demonstrated with dynamic MRI that androgen deprivation led to vascular collapse in prostate tumor [et al. suggested that hormonal agents may increase the percentage of normal vessels by inhibiting angiogenesis in hypervascular tumors, which results in oxygenation and thereby improvement in radiosensitivity [Movsas and Milosevic currence , 15. White tumor . Jain etsitivity . It was sitivity –17.Reducing tumor volume at the time of irradiation is considered to improve the effectiveness of radiotherapy, even if high doses are administered using HDR in our study. These things suggest that patients with a better response to NHT have shown more favorable prognosis regardless of the values of i-PSA. These results also correspond with the results of Zietman's report .et al. reported that high-risk prostate cancer  was a more important prognostic factor for distant metastasis than other factors, and local recurrence was the most important prognostic factor for distant metastasis [Coen tastasis . It is hIn radiotherapy for high-risk localized prostate cancer, concomitant long-term AHT has become standard treatment, but adequate dose escalation for the prostate and seminal vesicles as well as pre-treatment minimization of prostate cancer proliferation and tumor burden may be the most important thing, even if long-term AHT is combined.In high-risk prostate cancer patients treated with the trimodality therapy (being composed of various treatments), our results confirmed the importance of the dose of HDR and the pre-HDR PSA nadir as prognostic factors. Especially, pre-HDR nadir PSA values of ≤0.1 ng/ml after NHT were related to improved bRFS. Further studies are needed to confirm the relevance of pre-HDR PSA nadir in trimodality therapy.This work was supported in part by a grant of SHIP0804 (NCT00664456).This work was supported in part by a grant of Foundation for Biomedical Research and Innovation (NCT00664456).